Targeting the early phase of HIV-1 infection, including virus entry, as a prophylactic modality is a focus of intense research. HIV-1 entry involves a series of events that include attachment to the host cell and fusion of the viral and target cell membranes. HIV-1 entry is mediated by the viral spike, which is composed of three gp120 envelope glycoproteins and three gp41 transmembrane molecules. In humans, HIV-1 infection begins with two consecutive gp120 binding events, each associated with major conformational changes in the gp120 structure. The first involves gp120 binding to the host CD4 receptor. CD4 binding results in a major gp120 conformational change, thus exposing a site for binding to the chemokine receptor (either CCR5 or CXCR4). Chemokine receptor binding is accompanied by gp41 rearrangement and the insertion of the gp41 fusion peptide into the host cell membrane, permitting fusion and viral entry. The highly conserved gp120-CD4 interface has been revealed by a number of X-ray crystal structures of the gp120 core domain, complexed to the D1D2 fragment of CD4 and a Fab of a human neutralizing antibody 17b, the latter serving as a surrogate for the co-receptors. CD4 binding induces the formation of a large internal cavity at the interface of the three gp120 domains, the inner domain, the outer domain, and the bridging sheet domain. The Phe43CD4 and Arg59CD4 residues have been shown by both mutagenesis and structural studies to be critical for binding of gp120 to CD4. Residue Phe43CD4 is located on the CD4 CDR2-like loop and binds at the vestibule of the large cavity formed upon the CD4-induced gp120 conformational change; Arg59CD4 is located on a neighboring β-strand and forms an electrostatic interaction with Asp368gp120 at the cavity vestibule. The structure of the unbound form of the simian immunodeficiency virus (SIV) gp120, which has a 35% sequence identity with HIV-1 gp120, indicates an invariant outer domain, with conformational changes occurring in both the bridging sheet and inner domain. Recent studies indicate that the HIV-1 gp120 core exhibits a propensity to assume the CD4-bound conformation, but is restrained from doing so by gp120 variable loops and interactions with gp41 in the context of the trimer spike. The thermodynamic signature of the CD4-induced gp120 conformational change exhibits a highly favorable binding enthalpy balanced with a highly unfavorable entropy associated with molecular ordering.
Two N-phenyl-N′-(2,2,6,6,-tetramethyl-piperidin-4-yl)-oxalamide compounds, NBD-556 and NBD-557 (FIG. 7), were identified via screening a drug-like small-molecule library for inhibition of gp120-CD4 binding. Zhao, Q. et al. Virology 339, 213-25 (2005). The NBD chemotype is defined by three pharmacophores: Region I, a para-halogen substituted phenyl ring; Region II, an oxalamide linker, and Region III, a substituted piperidine ring (FIG. 7). Mutagenesis, modeling and synthesis of NBD analogues with improved binding affinity revealed that these small molecules bind to the highly conserved gp120 cavity and compete with CD4 binding. Schön, A. et al. Biochemistry 45, 10973-80 (2006); Schön, A. et al. Chem Biol Drug Des: 77, 161-165 (2011); Madani, N. et al. Structure 16, 1689-701 (2008); LaLonde, J. M. et al. Bioorganic & Medicinal Chemistry 19, 91-101 (2011). Exploration of structure-activity relationships (SAR) in Region III demonstrated that compounds with comparable binding affinities act both as CD4 antagonists (i.e., to inhibit HIV-1-infection of CD4+ cells) and as CD4 agonists (i.e., promote CCR5 binding and enhance viral infection in the absence of CD4). Madani, N. et al. Structure 16, 1689-701 (2008); LaLonde, J. M. et al. Bioorganic & Medicinal Chemistry 19, 91-101 (2011). Mimicry of CD4 was further demonstrated by the similarity of the NBD and CD4 thermodynamic signatures, both exhibiting a large unfavorable entropy change, −TΔS, to Gibbs energy (17.1 kcal/mol and 24.1 kcal/mol for NBD-556 and CD4, respectively) compensated by a large favorable enthalpy change (−24.5 kcal/mol and −34.5 kcal/mol for NBD-556 and CD4, respectively). Taken together, these results provided a rationale for further optimization of NBD analogues as inhibitors of HIV-1 viral entry by focusing on both Phe43 cavity and Asp368gp120 hotspots.
While structure-activity relationships have been explored extensively, the current lead compound, TS-II-224 (2) (FIG. 7) has a binding affinity of 0.33 μM with an IC50=89.9 μM. Modeling and subsequent crystal structures of TS-II-224 (2) and NBD-556 in complex with Glade C1086 gp120 verified that the NBD compounds bind in the Phe43 cavity. Moreover, the crystal structures reveal that the Region III tetramethylpiperidine interactions are dominated by van der Waals contacts rather than specific polar protein-ligand interactions. Thus, an essential component of the gp120-CD4 hotspot, the Asp368gp120-Arg59CD4 electrostatic interaction has not been successfully integrated into NBD small-molecule design. Previously, these features were incorporated in a small-molecule scaffold and a cyclic peptide. However, the problem remains refractory, as the spatial arrangement between the NBD Region II stem and Asp368gp120 is near 90 degrees, a trajectory difficult to capture in small-molecule scaffolds.
There exists a need for small molecule inhibitors that mimic the crucial Asp368gp 120-Arg59CD4 interaction at the dual gp120-CD4 hotspots. In certain embodiments, these CD4-mimetic compounds exhibit improved thermodynamic and antiviral properties.